This project is designed to continue an exploration of early responses to photodynamic therapy (PDT), a procedure that involves irradiation of photosensitized cells and tissues. PDT circumvents many common drug- resistance mechanisms encountered in cancer chemotherapy and can lead to effective cancer control. PDT was initially (ca. 1975) thought to kill cells via membrane photodamage. We now know that the process is much more complex, with both apoptosis and autophagy plahying important roles. In this proposal we have identified three aims. We propose to: [1] explore the role of autophagy as a factor in cell death vs. survival in PDT; [2] characterize the effects of PDT on the endocytic pathway; [3] assess the contributions and roles of different reactive oxygen species on initiation of apoptosis and autophagy. Studies in the 1990s established apoptosis as a common mechanism of photokilling but not until 2006 was it realized that autophagy was also a component of survival vs. death decisions. Our knowledge of PDT responses continues to evolve and now includes such determinants of efficacy as canonical vs. non-canonical routes to autophagy and the evocation of apoptosis via indirect routes. While apoptosis is an irreversible route to death, autophagy can serves as a survival pathway, introducing a 'shoulder' on the dose-response curve, or as a death pathway. The result appears to depends on the PDT target, intrinsic cellular capacity for apoptosis and autophagy and other characteristics of the cell phenotype. An additional factor may be the pathway to autophagy elicited by PDT: canonical vs non-canonical. The latter is said to lead to cell death, but this may represent a special case. Protection from photokilling can occur even when autophagy is incomplete, i.e., without the fusion of auto- phagosomes with lysosomes followed by proteolysis of the encapsulated cargo. The nature of reactive oxygen species (ROS) induced by PDT also plays a role in promotion of apoptosis and autophagy. Enhanced H O 2 2 production was found to lead to the promotion of apoptosis, an effect we attribute to the long persistence of this species in the cellular environment. Moreover, both H O and !O S have been suggested as potential 2 2 2 initiating factors for autophagy. An exploration of the role of different ROS as determinants of PDT efficacy is therefore proposed. We have recently found that PDT evokes a hitherto unreported cellular response: impaired membrane trafficking leading to interference with the endocytic pathway. The implications of this effect remain to be explored.